Methotrexate and other antifolates such as trimetrexate have been demonstrated to be effective chemotherapeutic agents in the treatment of a variety of human tumors. However, toxicity for normal hematopoietic and gastrointestinal tissues limits the usefulness of antifolates as antitumor agents. In studies conducted as a part of this project, we have demonstrated a quantitative increase in dose tolerance resulting from transplantation with donor marrow expressing a mutant dihydrofolate reductase (DHFR) enzyme, which is resistant to antifolates. Furthermore, we have also demonstrated that this increased dose tolerance brought about by drug-resistant DHFR expression can be used for improved chemotherapy against several different murine tumors. Based on the success of these studies in the murine model system, we now propose experiments that will contribute substantially to the translation of this approach to human clinical application. In Aim 1, lentivirus vectors will be developed as an effective system for transduction of the murine tyrosine-22 DHFR gene, conferring resistance to MTX and other antifolates. These vectors will be tested by exposure of normal mouse bone marrow cells with subsequent transplant into recipient animals and evaluation for increased MTX dose tolerance. In Aim 2, the DHFR lentivirus vectors will be tested for the ability to confer increased MTX dose tolerance in dogs as a large animal model system. Dogs transplanted with DHFR virus-transduced marrow will undergo a careful dose escalation regimen to determine the maximal amount of MTX tolerated without causing myelosuppression. In Aim 3, multi- potent adult progenitor cells (MAPC) will be evaluated in the murine system as a potentially superior target for DHFR gene transfer with the capability of contributing drug-resistant cells to both hematopoietic and gastrointestinal tissues. These studies are intended to provide significant preclinical results supporting the use of DHFR gene transfer for chemoprotection and improved antitumor chemotherapy in human clinical trials.